The present invention relates to a method and apparatus for the preparation of stained cells or cell objects on microscope slides for later use in image analysis or the like.
The variation in degree of staining cell objects and the non-uniformity of the staining of the cell objects on a cell slide presents a problem in the quantification of the stained cell objects by a subsequent image analysis technique. The term "cell objects" as used herein includes not only cells and cellular material such as DNA, but also includes artificial materials such as beads or the like which may be stained and used as a calibration material. The uniformity of staining may not be as important for large or gross evaluations of cellular material on a slide but can be critical in analysis of small cell objects, such as DNA within a cell nucleus. The cell objects are incredibly small for example, 100 micrometers.sup.2 in size or less. In such analysis, even small shifts in light transmission due to subtle staining variations can cause particular changes in a later diagnosis or prognosis. These subtle staining shifts may be much too subtle for the human eye; or so small that the human eye and a manual analysis of the cell objects is not affected. That is, the staining technique which is suitable and adequate for the human eye may not be adequate where there is a system using automatic analysis, which makes much finer determinations of grey values when, e.g., determining the absolute value of DNA content of a cell nuclei.
U.S. patent application Ser. No. 076,685 filed July 2, 1987 now abandoned and Ser. No. 121,674 filed Nov. 17, 1987 now U.S. Pat. No. 5,016,283 disclose staining kits which use a number of different staining stains and staining techniques and particularly, Feulgen staining technique to use to stain DNA in cell objects with dyes such as for example, with Thionin on cell objects such as rat liver cells. In addition, as disclosed in other patents and applications directed to this image analysis technique, there is often a need to analyze the morphological features such as the texture in combination with the size and shape of the cell nuclei and/or alterations in the nuclear cytoplasmic ratios of cells, all of which are dependent upon an accurate and uniform staining.
There are a number of available staining techniques which can be used. The Feulgen staining technique may be used to stain DNA in cell objects with dyes, for example, with Thionin, Azure A, Azure C, pararosanilin and methylene blue. Proteins may be stained with congo red, eosin, an eosin/hematoxylin combination, or fast green. Enzymes may be made visible with diaminobenzidine or 3-amino-9 ethylcarbazole or alkaline phosphatase in combination with a dye substrate; cell organelles may be stained with methylene blue; and ribosomes with methylene blue and mitochrondia with giemsa stain. Moreover, as used herein, stain includes counter stains such as methyl green. In breast cell cancer analysis some of these stains are used in combination with monoclonal antibodies which detect estrogen receptors. Antigen analysis may include the steps of binding of monoclonal antibodies to the specimen and control cell objects. Later the monoclonal antibody may be conjugated with an enzyme stain. Also, the monoclonal antibody may be conjugated with a fluorescent material. Then the fluorescent stain may be excited at a wavelength to induce the fluorescence and then this may be observed at another wavelength at which fluorescent emission occurs. When the antibody is made for a particular virus, the control cell specimen objects may be treated with a nucleic acid probe specific for the genome of the virus.
As disclosed in another application Ser. No. 121,674 filed Nov. 17, 1987, the staining of the cell population may include staining with an alkaline phosphatase technique using a monoclonal antibody against a specific cytoplasmic antigen. The resulting stain is substantially specific to the cytoplasm and does not stain the nucleus of the cells. A Feulgen staining process using Thionin is then performed to stain the DNA in the nucleus of each cell. The alkaline phosphatase staining method is used because of its compatibility with the Feulgen staining technique. The alkaline phosphatase staining is specific to the cytoplasmic antigen binding the chosen monoclonal antibody and does not harm the nuclear material so that it may receive the Feulgen stain in the subsequent step. The alkaline phosphatase staining is accomplished first before the destruction of the cytoplasm by the Feulgen staining technique. The chromogen chosen for the staining technique is a fast red dye which is advantageous for two reasons. In the first instance, the fast red dye which is precipitated is not susceptible to being washed out by the Feulgen staining process; and thus will remain for the optical visualization. The second reason is that the chromogen provides an excellent optical separation from the blue Thionin dye used in the Feulgen staining process.
Manifestly, there are other dyes and stains other than those listed and described in the aforesaid techniques wherein the staining of cell objects on slides is later used in analysis techniques such as image analysis. The present invention is not to be limited to the particular dyes or stains described above or the particular analysis used or described herein or described in the aforesaid patent applications, each of which is hereby incorporated by reference as if fully reproduced herein. Rather, the present invention is directed to the providing a more uniform staining technique for cell objects on microscope slides wherein staining uniformity is needed such as where there is an absolute value measurement of DNA or measurements of small cell areas or cellular masses in picograms.
In order to obtain the accuracy of measurement desired, the slides may be provided with calibration cell objects and then specimen cell objects to be analyzed are added onto the same slide. Both the calibration cell objects and the specimen cell objects are stained simultaneously; and then the image analysis apparatus is calibrated by comparing the stain on the calibration cell objects to a predetermined known standard, and adjustments are made for the staining deviation from the standard. Such slides having calibration cell objects thereon have been inserted into standard staining containers such as Coplin staining jars having grooves to hold a plurality of vertical slides back-to-back in the jar. Despite care taken in the mixing of the stain in the Coplin jars, it has been found that some stains, such as the Thionin stain, do not provide the same stain intensity. For example, the cells may not be stained the same amount even though they are in the same jar. This results in quality control problems. It has been found that the Thionin dyes are not very soluble in water and that the Thionin dyes sometimes tend to separate into different phases or levels with different stain concentrations in different levels. This may not be not apparent to the naked eye, but this appears, in fact, to be true. It has been found that if one turns several of the slides to have their calibration cells on their upper ends in the Coplin jar, that these calibration cells may have a different stain concentration from the calibration cells on the lower ends of other slides in the same Coplin jar. The slides are usually glass slides and may be easily broken; and the size of the slides and the Coplin jar as well as the amount of stain and the staining techniques have already been developed. Hence, it is desired to continue to use the same slides, stains and Coplin jars but to improve the uniformity of stain concentration in an inexpensive and simple manner.
Accordingly, a general object of the invention is to provide an apparatus and method for improving the uniformity of staining of cell objects on microscope slides.